The massive application of live-attenuated vaccines offer several economic and health inconveniences. When live vaccines are attenuated their immunogenic capability is often reduced. See Leclerc, et al., Immunol. Today, 19(7):300-302, (1998); Nieba, et al., Mod. Asp. Immunobiol., 1(2):36-39 (2000). Another inconvenience is the possibility that the attenuation be reverted and that the microorganism regain its disease inducing properties. See Redfield, N., N. Eng. J. Med., 316:673-678 (1998). Hence, during the past years, the trend has been to formulate acellular vaccines, based on individual compounds isolated from bacteria or virus. In general, these individual compounds, like proteins typical of a microorganism, have low immunogenicity. This limitation has been overcome by using adjuvant substances. However, there are proteins that even in the presence of adjuvant substances continue showing low immunogenicity. Several protein engineering strategies have been proposed to overcome these difficulties. See Leclerc, et al., Immunol. Today, 19(7):300-302 (1998).
Viral capsid proteins are able to form tridimensionally ordered particles, called “virus-like particles”. These particles have the same size and shape as whole viruses. However, they are empty inside and without genetic material rendering then incapable of producing infections. Their great size and order provide them with a marked immunogenicity. See Bachmann, et al., Science, 262:1448-1451 (1993). The recombinant vaccine against hepatitis B, widely accepted in the market, is based on this concept. The “virus-like particles” have been used as a vehicle for inserting peptides characteristic of certain pathogens with the aim of producing vaccines against such pathogenic microorganisms. See WO0032227 (Renner, et al.); WO0185208 (Bachmann, et al.). A favored strategy has been the insertion of multiple copies of a peptide in a very immunogenic vehicle, in order to provide a peptide with the adjuvant property of the carrier. However, this approach has encountered many difficulties: owing to the huge size of these particles, any insertion of a peptide in its compounding protein obstructs its proper folding and, in many cases, decreases its stability. Moreover, there are few sites able to accept peptide insertions without changing their general structure. See Nieba, et al. Mod. Asp. Immunobiol, 1(2):36-39 (2000).
Some bacterial proteins have been postulated as vehicles for developing chimeric vaccines. See Leclerc, et al., Immunol. Today, 19(7):300-302 (1998). The subunit B of the cholera toxin is a stable pentameric protein that has been used to obtain an immune response from the mucosa against inserted peptides. This strategy has been successful due to this toxin capacity to penetrate the gastric mucosa. See Arakawa, et al. Nature Biotech., 16:934-938 (1998). The dihydrolipoyl dehydrogenase enzyme of the Bacillus steearothermophilus has also been postulated as a proteic vehicle because it forms a complex and very stable polymeric structure. See Domingo, et al., J. Mol. Biol., 305:259-267 (2001); WO0142439 (Domingo, et al.).
The lumazine synthase enzyme catalyzes the penultimate step in the riboflavin biosynthetic route. See Goldbaum, et al., J. Med. Microbiol., 48:833-839 (1999). Its active site is located in the interphase among monomers, making this protein to have a very stable polymeric order. See Ritsert, et al. J. Mol. Biol., 253:151-167 (1995). These orders vary between proteins forming pentameric and icosahedric particles. See Braden, et al., J. Mol. Biol., 297:1031-1036 (2000). The icosahedric structure of the lumazine synthase of B. subtilis has been postulated as a vehicle for inserting peptides and developing vaccines. See WO0053229 (Bacher, et al.).
The lumazine synthase of Brucella spp. is a highly stable protein. It has been demonstrated that this 18-kDa protein is a useful marker for the serological diagnosis of human and animal brucellosis. See Goldbaum, et al., J. Clin. Microbiol., 30:604-607 (1992); Goldbaum, et al., J. Clin. Microbiol., 31:2141-2145 (1993); Baldi, et al., Clin. Diag. Lab. Immunol., 3 (4):472-476 (1996). According to immunochemical enzymatic function and tridimensional structure by X-ray crystallography analyses the original and modified protein shows the same when expressed recombinantly as the native protein. See Braden, et al. J. Mol. Biol., 297:1031-1036 (2000); Goldbaum, et al., J. Med. Microbiol., 48:833-839 (1999); Goldbaum, et al., J. Struct. Biol., 123:175-178 (1998). The structure shows that this 18-kDa protein behaves as a 180-kDa decamer in solution, becoming a new type of quaternary arrangement of the lumazine synthase. See Zylberman, et al., J. Biol. Chem., 279(9):8093-8101 (2004).
It has been postulated that the immunogenicity of the lumazine synthase of Brucella spp. derives mainly from its polymeric character. See Baldi, et al., Braz. J. Med. Biol. Res., 33:741-747 (2000). The structure also shows that the amino termini end of 10 aminoacids is involved neither in the general folding nor in the contacts among monomers. See Braden, et al. J. Mol. Biol., 297:1031-1036 (2000). The lumazine synthase of Brucella spp. is a powerful immunogen capable of producing a high humoral and cellular immune response in the murine model. This capability has been verified when the immunization is induced with the recombinant and modified protein and with a plasmid that codifies for the protein (gene therapy, DNA vaccination). See Velikovsky, et al., J. Immunol. Meth., 244(1-2):1-7 (2000). It is possible to modulate the response by changing the immunization route and the adjuvant used. See Velikovsky, et al., Infec. Immun., 70(5):2507-11 (2002). Particularly, it is possible to create a strong response of the TH1 type, which would be the response with the highest protecting capacity in brucellosis. See Velikovsky, et al. Infec. Immun., 70(5):2507-11 (2002).
However, it is still a need in the art for new vehicles with smaller stable structures useful for displaying peptides, polypeptides and proteins, in general, and antigens or immune response-inducing agents, in particular. The development and use of the lumazine synthase decameric structure as a vector of this type have not been described in the art.